2, 2&#39;-succinyl bis



United States Patent 3,352,881 2,2'-SUCCiNYL BllS(4-THIAZOLECAR- BOXYLIC ACID) Miklos Bodanszky, Princeton, and John T. Sheehan, Middlesex, N.J., assignors, by mesne assignments, to E. R. Squibb & Sons, Inc., New York, N.Y., a corporation of Delaware No Drawing. Original application Mar. 4, 1964, Ser. No.

355,142, now Patent No. 331,864, which in turn is a division of. application Ser. No. 206,155, June 29, 1962, now Patent No. 3,201,457, which in turn is a division of application Ser. No. 137,495, Sept. 12, 1961, now Patent No. 3,065,240, which in turn is a division of application Ser. No. 47,129, Aug. 3, 1960, now Patent No. 3,040,054, dated June 19, 1962. Divided and this application Feb. 2, 1967, Ser. No. 641,388

1 Claim. (Cl. 260--302) ABSTRACT OF THE DISCLOSURE This application is directed to 2,2'-succinyl his(4-thiazole carboxylic acid), which is useful as a precursor of the antibiotic thiostrepton.

This application is a division of our application, Serial No. 355,142, now US. Patent No. 3,331,864, filed March 4, 1964, which in turn is a division of our application, Serial No. 206,155, filed June 29, 1962, now Patent No. 3,201,457, which in turn is a division of our application, Serial No. 137,495, filed September 12, 1961, now Patent No. 3,065,240, which in turn is a division of our application, Serial No. 47,129, filed August 3, 1960, and now Patent No. 3,040,054, granted June 19, 1962.

This invention relates to new chemical compounds and more particularly to a new thiazolecarboxylic acid of the formula 3 l I S j n-onomomonwfl HOOC N N 000151 and salts thereof. Since the thiazolecarboxylic acid contains both basic (i.e. amino) groups and carboxylic acid groups it is amphoteric in nature and thus forms salts with acids and bases. Although any acid or base may be used in preparing the salts, the preferred acids are mineral acids, such as the hydrohalic acids (e.g., hydrochloric and hydrobromic acid), sulfuric acid, nitric acid and phosphoric acid. The preferred bases are ammonium hydroxide, alkali metal hydroxides (e.g., potassium hydroxide and sodium hydroxide) and alkaline earth metal hydroxides (e.g., calcium hydroxide). In addition organic acids such as tartaric, citric and acetic acid, and organic bases, such as alkylamines, can be used.

The new thiazolecarboxylic acid of this invention (as well as its salts) is useful as a precursor in the microbial preparation of the antibiotic thiostrepton. For this purpose it is added at a concentration greater than about 0.001% (W./v.) to a nutrient medium containing a source of carbon and energy, a source of assimilable nitrogen and a source of assimilable sulfur (such as that described in US. Patent No. 2,982,689) when culturing Streptomyces aureus (Waksrnan Collection No. 3705, Institute of Microbiology, New Brunswick, NJ.) for the production of thiostrepton.

The final products of this invention are prepared by a series of reactions using as the initial reactant succinaldehyde. These reactions are shown schematically by the following equations. In these equations, R is a hydrocarbon radical of less than ten carbon atoms, such as lower alkyl (e.g., methyl, ethyl, propyl, butyl and hexyl),

aryl (e.g., phenyl, p-tolyl and o,p-xylyl), aralkyl (e.g., u-tolyl, phenethyl and Z-phenylpropyl), and cycloalkyl (e.g., cyclopentyl and cyclohexyl); and R' is a saturated hydrocarbon radical of less than ten carbon atoms, such as lower alkyl (e.g., methyl, ethyl, propyl and butyl). Each of the intermediates formed is a new compound of this invention. -In addition to their use as intermediates, Compounds IV and V can also be used as precursors in the production of thiostrepton.

KCN oonornoniono RCOX OI (aoono ON CN 1 His noooorronicmonooon I R=phenyl I l ll C-NH: O-NHa x'omoocoon' Rcooonomonzdnoooa II R=phenyl noooorromonionoooa /S\ Eh n KOH ROOC-CN rlo-ooon' 2 III R=phenyl; R=CzH on on S I l S N32Cl101 J Tornomomon-Wl HOOC *N N 00011 0 0 s H H s HoooNrn j To-omcrno-H l H000 N N 00011 NH, NH, s s j Tonotncm-on 1 H000 -N N 00011 To prepare Compounds A, succinaldehyde is interacted with a cyanide, such as an alkali metal cyanide (e.g., potassium cyanide), and an acylating agent of either the formula RCOX or (RCO) O, wherein R is as hereinbefore defined, and X is a halide, preferably chloride. The reaction is conducted in an aqueous medium preferably in the cold, and results in the preparation of the two isomers of the diesters of 2,5-dihydroxyhexanedinitrile. Compounds A, the acid moiety of the ester corresponding to the acylating agent used.

To prepare Compounds B, either of the isomers of Compounds A is treated with hydrogen sulfide, the reaction preferably being conducted under basic conditions. The reaction results in the preparation of Compounds B, the diesters of 2,5-dihydroxydithiohexanediamide, wherein the acid moiety of the ester corresponds to the acylating agent used in the first step of the process.

Compounds B are then condensed with an ester of halopyruvic acid of the formula: XCH COCOOR', wherein X is halo, preferably bromo, and R is as hereinbefore defined. The reaction is preferably conducted in an organic solvent at an elevated temperature, such as the reflux temperature of the solvent, and results in the preparation of pound IV, 2,2'-(1,4-dihydroxytetramethylene)bis-(4-thi-.

azolecarboxylic acid).

Compound IV is then oxidized by treatment with sodium dichromate to give Compound V, 2,2'-succinylbis(4- thiazolecarboxylic acid).

Compound V is then treated with ammonium formate at an elevated temperature and the resulting formyl derivative hydrolyzed, as by treatment with a mineral acid to yield one of the final compounds of this invention, Compound VI, 2,2-(l,4-diarninotetramethylene)bis(4- thiazolecarboxylic acid) which can be converted to a salt in the usual manner by treatment with the desired acid or base.

The following examples illustrate the processes of this invention (all temperatures being in centigrade):

Example 1.-Benzic acid, diester with 2,5-dihydr0xyhexanedinitrile (I) A mixture of 30 g. of succinaldehyde, 750 g. of cracked ice, 44 g. of potassium cyanide and 94 g. (78 ml.) of benzoyl chloride is stirred for 1 hour. A milky semi-solid separates from the reaction mixture. The mixture is filtered by suction, and the white solid residue washed with water to give on drying about 15 g. Solid 1. The original filtrate of the reaction mixture is extracted four times with 250 ml. portions of chloroform and the combined chloroform extract washed first with a solution containing 60 g. of sodium bicarbonate in 750 ml. of water and finally with three portions of 250 ml. of water. The chloroform extract is dried over anhydrous magnesium sulfate and after removing the latter and evaporating the solvent a residue of about 62 g. of heavy liquid remained. This residue on treatment with several volumes of hexane gives about 19 g. of Solid II.

Solids I and II are separately treated with ether, from which extracts benzoic acid is recovered. The ether .in-

soluble materials are treated with toluene on the steam bath and from these extracts a material, M.P. about 134-136", separates. I

Analysis.-Calcd for C I-I N O C, 68.96; H, 4.63; N, 8.04. Found: C, 68.89; H, 4.60; N, 7.93.

The toluene materials are then treated with dimethylformamide on the steam bath and solution is complete. On cooling a crystalline product melting at about 214- 216 separates.

Analysis.-Found: C, 68.96; H, 4.81; N, 8.10.

The infrared spectra of both compounds are substantially the same.

Example 2.-Benzoic acid, diester with 2,5-diliya'r0xydithiohexanediamide (II) Into a solution of 4.6 g. of the lower melting dinitrile obtained in Example 1 in 25 'ml. of dimethylformamide containing 0.3 ml. of triethanolamine is bubbled hydrogen sulfide for six hours. After the addition of gas, the stoppered mixture is allowed to stand for 24 hours. The mixture is then treated with hexane and enough ethanol to make the mixture homogeneous. On standing 9. yellow solid slowly separates. Ultimately with scratching and further diluting with hexane and ethanol about 5 g. of product is obtained. The product is crystallized from large volumes of boiling ethanol and melts at about 203-205.

Analysis.-Calcd for C H N Q S N, 6.73; S, 15.37. Found: N, 6.94; S, 15.40.

Example 3.--2,2'(I,4 dihydroxytetrametlzylene)bis(4 thiazolecarboxylic acid) dietllyl ester, diestel' with bonzoz'c acid (III) A mixture of 2.5 g. of the dithioamide obtained in Example 2 and 2.5 g. of ethyl bromopyruvate in 100 ml. of ethanol is heated under reflux for one and one-half hours on the steam bath. On cooling a solid separates. The solid is filtered off to yield about 2.9 g. of solid, which is crystallized from ml. of boiling ethanol. The product melts at about 144-145.

Analysis.Calcd for C30H28N2OBS2: N, 8,1052. Found: N, 4.68; S, 10.61.

Example 4.2,2'-(1,4 dihydroxytetramethylene)bis(4 thiazolecarboxylic acid) (IV) 2.1 g. of the ester obtained in Example 3 is suspended in 50 ml. of absolute ethanol and 0.76 g. of potassium hydroxide in 5 ml. of absolute ethanol is added. While stirring at room temperature solution occurs within onehalf hour. The mixture is allowed to stand at room temperature overnight during which time a solid separates out. The insoluble material is filtered off and washed with ethanol. It is dissolved in 10 ml. of water and the aqueous solution then washed with ether and then acidified with 1 N hydrochloric acid. From the acid solution a precipitate slowly formed which is filtered off, washed with water, and air dried to give about 1.0 g. of product, M.P. about 235-240. From the original mother liquor on evaporation a residue of about 1.6 g. of product is obtained. The residue is dissolved in 10 ml. of water and after washing the aqueous solution with ether, the aqueous solution is acidified with hydrochloric acid and the precipitate which forms filtered off. This material is 'benzoic acid. From this and the previous acid solution however, on cooling and standing about 0.2 g. additional product separates. Crystallized from boiling water, this compound separates out with one mole of water of crystallization which is lost on drying at The compound, 2,2-(1,4-dihydroxytetramethylene)bis(4-thiazolecarboxylic acid melts at about 240242 (dec.).

Analysis.-Calcd for C H N O S C, 41.87; H, 3.51. Found: C, 42.01; H, 3.95.

Example 5.--2,2'-succinyl bis (4-thiaz0lecarb0xylic acid) (V) 600 mg. of the diol acid obtained in Example 4 is sus pended in 15 ml. of glacial acetic acid distilled from chromium trioxide, and 500 mg. of sodium dichromate in one-half a cubic'centimeter of water is added to the suspensiomThe whole is kept at 100 for 2 hours. On cooling an insoluble material settles out. This material is filtered off and washed with a large amount of water and some dilute acetic acid. It weighs about 400 mg. After crystallization from dimethylformamide the 2,2-succinyl bis(4- thiazolecarboxylic acid) has a M.P. of about 284286 (dec.).

Found: C, 42.52; H, 2.90.

Example 6.-2,2- (1,4-diamin0tetramethylene bis-4- (thiazolecarboxylic acid) (VI) 100 mg. of the diketo. acid obtained in Example 5 is intimately mixed with 2.0 g. of ammonium formate in a Claisen type distilling flask. The flask is heated in an oil bath at an internal temperature of -155 until. the initial foaming reaction subsides. The internal temperature is then gradually raised to -l90 where it is kept for3 hours. On cooling the residue is taken up in 25 ml. of water and any insoluble material removed via filtration. The filtrate is acidified with hydrochloric acid whereupon a precipitate separates out. It is filtered 0E and washed with water to yield, a mixture offormyl derivatives. This mixture is heated under reflux with 5 m1. of concentrated hydrochloric acid for an hour. Hydrolysis proceeds and the mixture becomes homogeneous. On evaporation the residue obtained is dissolved in water from which VI slowly separates. It melts at about 235-237" (dec). Calcd for C12H14O4N4S2: C, H, N, S, 18.7. Found: C, 42.5; H, 4.29; N, 16.4; S, 18.8. On Whatman No. 1 paper in;a system of n-butanol-acetic acid-water (4:121) the acid moves faster than cystine, RF 0.008- 0.10.

Example 7.-2,2' (1,4 diaminotetramethylene) bis(4 thiazolecarboxylic acid), dihydrochloride 40 mg. of 2,2 (1,4 diaminotetramethylene)bis(4 thiazolecarboxylic acid) is dissolved in warm N-hydrochloric acid. The dihydrochloride salt which forms slowly separates from solution and is removed by filtration.

Example 8.-2,2 (1,4 diaminole tramethylene)bis(4 thiazolecarboxylic acid), ammonium salt 30 mg. of 2,2 (1,4 diaminotetramethylene)bis(4 thiazolecarboxylic acid) is dissolved in an excess of am- 6 moniuin hydroxide. Evaporation of the solvent yields the ammonium salt.

The invention may be variously otherwise embodied within the scope of the appended claim.

What is claimed is: 2,2-succinyl bis(4-thiazolecarboxylic acid).

References Cited UNITED STATES PATENTS 6/1962 Bodanszky et a1. 260-302 ALTON D. ROLLINS, Primary Examiner. 

